Biopharmaceuticals are medical drugs produced using biotechnology. They include proteins (including antibodies), nucleic acids (DNA, RNA or antisense oligonucleotides) and living microorganisms like virus and bacteria where the virulence of viruses and bacteria is reduced by the process of attenuation. They can be used for therapeutic or in vivo diagnostic purposes, and are produced by means other than direct extraction from a native (non-engineered) biological source.
The removal of host cell impurities is a critical step in the production of biopharmaceutical products. One impurity targeted for clearance during the purification process is residual DNA arising from host cells. Traditional methods of quantitating residual host cell DNA have been limited by laborious sample preparation protocols, lack of sensitivity and specificity, and slow time to results.
In the process of manufacturing biopharmaceuticals, strict guidelines need to be followed to ensure the final pharmaceutical formulations are free of impurities and meet the regulatory guidelines. Among the common impurities are host cells which need to be removed before the final formulation. Majority of the therapeutic proteins are expressed using recombinant technology in the cell lines derived from the rodent family. The purified protein from the bulk harvest is likely to carry traces of CHO host cell genomic DNA into the final formulation thereby posing additional risk. Therefore, regulatory agencies like the world health organization (WHO), European Medicine Agency (EMA) or Food and Drug Administration (FDA) have documented the permissible levels of host cell DNA per dose of the drug administered to the patients in the guidelines. The present disclosure discloses a method of early detection and quantification of host cell genomic DNA contamination using a quantitative real time PCR.
In addition to potential safety issues stemming from the presence of extraneous host cell DNA, the regulatory guidance for products produced in cell culture is that DNA content in the final product should be as low as possible, as determined by a highly sensitive method. Traditional methods of quantifying levels of residual host cell DNA have been limited by lack of sensitivity and specificity, poor assay reliability, low sample capacity, slow time-to-results and high cost per sample tested.
Residual DNA in final bulk products should be generally lower than 100 pg per therapeutic dose (Points to consider in the manufacture and testing of monoclonal antibody products for human use, Centre for Biologics Evaluation and Research, US food and drug administration; 28 Feb., 1997; www.fda.gov/cber/gdlns/ptc_mab.pdf). The FDA also recommends that detection methods used be sensitive enough to detect residual amounts present in picogram levels. Three techniques have the required sensitivity to detect picogram levels of impurities: hybridization, methods based on DNA-binding protein and quantitative PCR (q-PCR).
Goldman et al. (Clinical chemistry 37:1523, 1991) describes a method of determining levels of DNA contamination using polymerase chain reaction. However, the methods described here are not sensitive enough to detect contamination in picogram levels. U.S. Pat. No. 5,393,657 discloses the use of primers and PCR amplification to detect the presence of host cell DNA contamination. However, conventional molecular biology techniques such as Southern blotting and gel electrophoresis are used for detection and quantification of the impurities. This reduces the overall ease of operation as well as the sensitivity.
U.S. Patent application No. 2009/0325175 discloses a method of identifying host cell genomic DNA contamination utilizing quantitative real time Polymerase chain reaction, wherein the qPCR primers are complementary to the highly repetitive host cell genomic DNA sequences, e.g. Alu-equivalent sequences. The cited document does not talk about the specific 74 bp Alu consensus sequence for which the instant disclosure become more sensitive. Therefore, a process which increases specificity and sensitivity in detecting the presence of host cell contamination is required. The present disclosure can detect the residual DNA impurities from the host cell of the rodent family as low as 20 femtogram in a recombinant protein product by using specific primers and probe.